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Journal of Virology, October 1999, p. 7988-7993, Vol. 73, No. 10
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Regulation of Closterovirus Gene Expression Examined by Insertion of a Self-Processing Reporter and by Northern Hybridization

Yuka Hagiwara,1 Valery V. Peremyslov,1 and Valerian V. Dolja1,2,*

Department of Botany and Plant Pathology1 and Center for Gene Research and Biotechnology,2 Oregon State University, Corvallis, Oregon 97331

Received 6 April 1999/Accepted 2 July 1999

A reporter open reading frame (ORF) coding for a fusion of bacterial beta -glucuronidase (GUS) with a proteinase domain (Pro) derived from tobacco etch potyvirus was utilized for tagging individual genes of beet yellows closterovirus (BYV). Insertion of this reporter ORF between the first and second codons of the BYV ORFs encoding the HSP70 homolog (HSP70h), a major capsid protein (CP), and a 20-kDa protein (p20) resulted in the expression of the processed GUS-Pro reporter from corresponding subgenomic RNAs. The high sensitivity of GUS assays permitted temporal analysis of reporter accumulation, revealing early expression from the HSP70h promoter, followed by the CP promoter and later the p20 promoter. The kinetics of transcription of the remaining BYV genes encoding a 64-kDa protein (p64), a minor capsid protein (CPm), and a 21-kDa protein (p21) were examined via Northern blot analysis. Taken together, the data indicated that the temporal regulation of BYV gene expression includes early (HSP70h, CPm, CP, and p21 promoters) and late (p64 and p20 promoters) phases. It was also demonstrated that the deletion of six viral genes that are nonessential for RNA amplification resulted in a dramatic increase in the level of transcription from one of the two remaining subgenomic promoters. Comparison with other positive-strand RNA viruses producing multiple subgenomic RNAs showed the uniqueness of the pattern of closterovirus transcriptional regulation.

* Corresponding author. Mailing address: Department of Botany and Plant Pathology, Oregon State University, Cordley Hall 2082, Corvallis, OR 97331. Phone: (541) 737-5472. Fax: (541) 737-3573. E-mail: doljav{at}bcc.orst.edu.

Journal of Virology, October 1999, p. 7988-7993, Vol. 73, No. 10
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Peng, C.-W., Napuli, A. J., Dolja, V. V. (2003). Leader Proteinase of Beet Yellows Virus Functions in Long-Distance Transport. J. Virol. 77: 2843-2849 [Abstract] [Full Text]  
  • Kreuze, J. F., Savenkov, E. I., Valkonen, J. P. T. (2002). Complete Genome Sequence and Analyses of the Subgenomic RNAs of Sweet Potato Chlorotic Stunt Virus Reveal Several New Features for the Genus Crinivirus. J. Virol. 76: 9260-9270 [Abstract] [Full Text]  
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